Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 295

and Halotag Ò technologies have been optimized so far under the name Tag-
lite Ò by Cisbio Bioassays. In the next paragraph, we will explain further the
assay based on the SNAP-tag Ò , as the principle is the same for the different
tags and only the nature of the substrate differs between the three labeling
methods.
The SNAP-tag Ò is a mutant of a DNA repair protein O 6 -alkylguanine
DNA alkyltransferase which was optimized to react preferentially to
benzylguanine derivatives. This reaction leads to irreversible covalent binding
of the probe to the SNAP-tag Ò and release of guanine. 47 In the Tag-lite Ò
binding assay, 19 the SNAP-tag Ò is fused to the N-terminus of a given GPCR
and the benzylguanine derivatized with a TR-FRET-compatible fluo-
rophore. This fusion is expressed in cell lines and labeled either with
SNAP-Lumi4Tb substrate, an HTRF-compatible FRET donor, or with a
SNAP-Red or SNAP-Green FRET acceptor. The cells can then be used im-
mediately or stored in liquid nitrogen for later use. To proceed with the bind-
ing assay, the labeled cell suspension is dispensed in the assay plate together
with the compound library to be screened, followed by the fluorescent ligand.
Once equilibrium is reached, the TR-FRET signal is measured directly in an
HTRF-compatible plate reader ( Fig. 7.3 ). Note that the assay is also applicable
on adherent cells and both with transiently transfected and stable cell lines.
Tag-lite Ò binding experiments have been performed on several GPCRs
including class-A b 2 adrenergic receptor, dopamine D2 receptor, opioid re-
ceptors, the class-B receptor VPAC1 for the vasoactive intestinal peptide,
and the class-C GABA B receptor. 19,51 When compared to values from
the literature, the K i values measured with the Tag-lite Ò assays were in
the same range and the ranking order of the compounds was similar. In
addition, the signal-to-noise ratio was found to be very high, and the
specificity of the signal good enough for analysis in the 384-well plate
format. A deeper analysis has been performed on the ghrelin receptor
GSH-R1a. 51 Using the Snap-tagged receptor and a red fluorescent
ghrelin ligand, 14 compounds were tested in a Tag-lite Ò binding
experiment. The same compounds, including agonists, antagonists, and
inverse agonists, were also tested in parallel in a radioactive binding assay.
This study showed a good correlation for the compound potencies
obtained in the two assays, and Tag-lite Ò binding assay was performed
both on adherent cells and using cell suspension. In addition, Tag-lite Ò
assay has been scaled down to the 384-well plate format, and the high
signal-to-noise ratio makes it compatible with 1536-well plates.